Influenza viruses (Orthomyxoviridae family) cause a highly contagious respiratory disease in humans resulting in approximately 36,000 deaths in the United States annually. The long term goal of this research is to reveal the mechanism by which the influenza non-structural protein-1 (NS1A) protein influences virus virulence utilizing in vitro, in vivo, and murine animal models. The hypothesis promoting this research is that the NS1A protein contains a previously uncharacterized site that participates in virus virulence. We propose that this region is a nuclear export signal (NES) and that inhibiting this NES enhances virulence of the virus by promoting NS1A nuclear functions. This hypothesis is based on observations that 1) an influenza A recombinant virus expressing an NS1 protein with a single alanine substitution at position L77 replicates 10-fold faster in a single cycle and 1000-fold faster in multiple cycles of replication, 2) this mutation in the NS1A protein leads to a substantially increased rate of virus-specific RNA synthesis, 3) recent protein sequence analysis places the nuclear export signal of NS1A in the virulence region identified above, and 4) the NS1A L77A mutation imparts increased pathogenicity to the virus in mice without prior adaptation (a characteristic previously attributed only to the viral hemagglutinin), demonstrating that this specific region of the NS1A protein significantly impacts the virulence of the virus. Based on these preliminary observations, the understanding of this functional site in the NS1A protein (hereafter termed the NS1A virulence region or NS1A VR) and the mechanism by which it influences virulence are the focal points of this proposal. The specific aims are to: 1. Define the boundaries of the NS1A VR that surround amino acid L77, by mutating the NS1A proteins of both avirulent and highly pathogenic strains. 2. Determine the effect of NS1A VR mutations on the nuclear-cytoplasmic shuttling of the NS1A protein. 3. Determine the mechanism of virulence by analyzing the gene and/or cytokine profile of infected tissues, virus tissue distribution and rate of replication, neutralizing antibody response, and/or leukocyte infiltration by comparing the wt and NS1A VR mutant viruses using cultured cells and a mouse animal model. [unreadable] [unreadable] [unreadable]